This R21 proposal aims to develop a versatile gene-trap approach for insertional mutagenesis in mouse embryonic stem (ES) cells. The specific applications of this approach include: 1) It will generate null mutations in trapped genes by transcriptional termination rather than protein truncation. 2) Expression of the trapped gene can be controlled spatially by tissue-specific repair of the null mutation following recombinase-mediated excision of the trap vector. 3) Expression of the trapped gene can be controlled temporally by a tetracycline-dependent promoter in the trap vector. 4) The expression pattern of the trapped gene can be easily assessed by B-galactosidase expression. 5) Genomic DNA of the trapped gene can be rapidly cloned and sequenced following plasmid rescue. 6) cDNA of the trapped gene with full-length coding sequence can be rapidly cloned and sequenced following 3' RACE. 7) The promoter of the trapped gene can be used to express any genes of interest following recombinase mediated cassette exchange in ES cells and replacement of the trap vector with the gene of interest. Specific aim 1 is to construct the trap vector. Aim 2 is to test all functional units in the trap vector in ES cells. Aim 3 is to test the above applications in mice derived from independent ES cell clones. The long-term goal of this project is to generate a C57BL/6 ES cell library and mutant mice that will permit spatial and temporal control of expression of trapped genes. They will provide valuable resources to the scientific community for the study of gene function in vivo.